Essay
Sex Determination: Why So Many Ways of Doing It?
Doris Bachtrog1*, Judith E. Mank2, Catherine L. Peichel3, Mark Kirkpatrick4, Sarah P. Otto5,
Tia-Lynn Ashman6, Matthew W. Hahn7, Jun Kitano8, Itay Mayrose9, Ray Ming10, Nicolas Perrin11,
Laura Ross12, Nicole Valenzuela13, Jana C. Vamosi14, The Tree of Sex Consortium"
1 University of California, Berkeley, Department of Integrative Biology, Berkeley, California, United States of America, 2 University College London, Department of Genetics,
Evolution and Environment, London, United Kingdom, 3 Fred Hutchinson Cancer Research Center, Divisions of Human Biology and Basic Sciences, Seattle, Washington,
United States of America, 4 University of Texas, Department of Integrative Biology, Austin, Texas, United States of America, 5 University of British Columbia, Department of
Zoology, Vancouver, British Columbia, Canada, 6 University of Pittsburgh, Department of Biological Sciences, Pittsburgh, Pennsylvania, United States of America, 7 Indiana
University, Department of Biology, Bloomington Indiana, United States of America, 8 National Institute of Genetics, Ecological Genetics Laboratory, Mishima, Shizuoka,
Japan, 9 Tel Aviv University, Department of Molecular Biology and Ecology of Plants, Tel Aviv, Israel, 10 University of Illinois, Department of Plant Biology, UrbanaChampaign, Illinois, United States of America, 11 University of Lausanne, Department of Ecology and Evolution, Lausanne, Switzerland, 12 University of Oxford,
Department of Zoology, Oxford, United Kingdom, 13 Iowa State University, Department of Ecology, Evolution and Organismal Biology, Ames, Iowa, United States of
America, 14 University of Calgary, Department of Biological Sciences, Calgary, Alberta, Canada
Abstract: Sexual reproduction is
an ancient feature of life on earth,
and the familiar X and Y chromosomes in humans and other model
species have led to the impression
that sex determination mechanisms are old and conserved. In
fact, males and females are determined by diverse mechanisms that
evolve rapidly in many taxa. Yet
this diversity in primary sex-determining signals is coupled with
conserved molecular pathways that
trigger male or female development. Conflicting selection on different parts of the genome and on
the two sexes may drive many of
these transitions, but few systems
with rapid turnover of sex determination mechanisms have been rigorously studied. Here we survey
our current understanding of how
and why sex determination evolves
in animals and plants and identify
important gaps in our knowledge
that present exciting research opportunities to characterize the evolutionary forces and molecular
pathways underlying the evolution
of sex determination.
variance that is otherwise hidden [2].
While many unicellular organisms produce gametes of equal size (isogamy, see
Box 1), sexual reproduction in most
multicellular organisms has led to the
evolution of female and male gametes
differing in size (anisogamy), and often to
the evolution of two separate sexes. Even
though the outcome of sex determination—whether an individual produces
relatively few large ova or many small
sperm—is strongly conserved, a bewildering number of underlying mechanisms can
trigger development as either a male or
female [3,4].
In humans, sex is determined by sex
chromosomes (XX females, XY males). The
X and Y chromosomes harbor dramatically
different numbers and sets of genes (about
1,000 genes on the X and only a few dozen
genes on the Y), yet they originated from
ordinary autosomes during the early evolution of mammals (Figure 1). Restriction of
recombination followed by gene loss on the
Y has resulted in the morphological differentiation of sex chromosomes (for a review
of the molecular and evolutionary processes
involved in Y degeneration, see [4,5]). The
vast majority of genes on the sex chromosomes are not directly involved in sex
determination, and development as a male
or female depends on the presence of a
single master sex-determining locus, the Sry
gene, on the male-limited Y chromosome.
Expression of Sry early in embryonic
development initiates testis differentiation
by activating male-specific developmental
networks, while in its absence, ovaries
develop. The first visible signs of sexual
differentiation of the ovary and testis occur
by the sixth week of gestation in humans
[6], and sex hormones initiate further sexual
differentiation in nongonadal tissues and
organs [7]. When this developmental process goes awry, the effects can be catastrophic, causing everything from ambiguous external genitalia (which occurs in up to
one in 4,500 infants) to sterility (which is
more cryptic and difficult to diagnose but
may be far more common).
Like humans and most mammals, other
genetic model systems, such as Drosophila
melanogaster flies and Caenorhabditis elegans
nematodes, harbor sex chromosomes, and
their commonalities have led to general
assumptions about the conservation of sex
determination mechanisms. However,
these model organisms present a false
impression of stability in how sex is
determined, and their commonalities
mask the diversity and turnover in sex
determination mechanisms that is readily
Introduction
Sex—the mixing of genomes via meiosis
and fusion of gametes—is nearly universal
to eukaryotic life and encompasses a
diverse array of systems and mechanisms
[1]. One major role of sex is to bring
together alleles carried by different
individuals, revealing beneficial genetic
Essays articulate a specific perspective on a topic of
broad interest to scientists.
Citation: Bachtrog D, Mank JE, Peichel CL, Kirkpatrick M, Otto SP, et al. (2014) Sex Determination: Why So Many
Ways of Doing It? PLoS Biol 12(7): e1001899. doi:10.1371/journal.pbio.1001899
Published July 1, 2014
Copyright: ß 2014 Bachtrog et al. This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium,
provided the original author and source are credited.
Funding: The Tree of Sex Consortium was funded by NESCent. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: dbachtrog@berkeley.edu
" Membership of the Tree of Sex Consortium is provided in the Acknowledgments.
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apparent when taking a broader taxonomic view. In this article, we address three
common myths about sex determination
and then deconstruct them based on a
broad taxonomic survey of animals and
plants.
Myths of Sex Determination
Myth 1: Sex is typically determined
by X and Y chromosomes
Many biologists are habituated to thinking about sex determination through the
familiar examples of mammals and D.
melanogaster, and assume that sex determination by sex chromosomes is the norm,
that males are XY and females are XX,
and that sex chromosomes are a stable
component of the genome. While biologists are generally aware of other modes of
sex determination (such as female heterogamety in birds, temperature-dependent
sex determination in reptiles, or development of males from unfertilized eggs in
bees), these alternatives are often viewed as
strange and aberrant [8].
Myth 2: Sex is controlled by one
master-switch gene
Sex determination in model species
suggests that a master-switch gene (e.g.
Sry in mammals, Sxl in D. melanogaster, and
xol-1 in C. elegans) acts as the main control
element to trigger either male or female
sexual development. Changes in the sex
determination pathways across taxa are
assumed to involve adding a new masterswitch gene to this molecular pathway (as
in some fly taxa; [9]), with little change to
downstream elements of the sex determination pathway [10]. A few genes are
thought to have the capacity to take on the
role of sex determination genes, and these
have been co-opted as master-switch genes
independently in different lineages (for
example, dmrt1 in several vertebrates
[11–14] and tra in insects [15–17]).
Myth 3: Sex chromosome
differentiation and degeneration is
inevitable
Sex chromosomes originate from identical autosomes by acquiring a sex determination gene (for example, the origin of
the Sry gene in mammals approximately
180 million years ago or Sxl in the
Drosophila genus .60 million years ago).
They are then thought to differentiate
through an inevitable and irreversible
process in which recombination between
X and Y chromosomes is shut down and
the Y degenerates (see Figure 1). Ultimately, Y chromosomes are fated to
disappear entirely (‘‘born to be destroyed,’’
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[18]). Thus, sex chromosomes that are
morphologically similar (homomorphic)
must be evolutionarily young, and in time
they too will degenerate.
While the evolution of anisogamy led to
the evolution of male and female functions, the evolution of separate sexes is not
inevitable across lineages. Indeed, most
flowering plants (94%, [19]) have both
male and female sex organs within a single
individual and often within the same
flower. By contrast, hermaphroditism is
rare among animals considered as a whole
(about 5% of all species), which is largely
due to the absence of hermaphrodites in
the species-rich insects, but it is common
in many other animal taxa, including fish
and many invertebrates (most snails,
corals, trematodes, barnacles, and many
echinoderms) [20]. Hermaphrodites can
mate with each other and benefit from the
advantages of sex by mixing their genomes, but when mates are difficult to
find, hermaphrodites can also escape the
need for a reproductive partner by selffertilization (which, however, may produce
low-fitness offspring due to ‘‘inbreeding
depression;’’ see below). This advantage of
reproductive assurance is particularly pronounced in sessile animals—like corals—
and plants, which cannot move to find a
mate [21,22]. Thus there is a clear
advantage to combining both male and
female functions within an individual,
especially in taxa with low mobility.
However, in some plants and most
animals, species are driven to separate
the sexes. This can be achieved in several
ways. One partial solution is the spatial
separation of male and female gonads in
the same individual, as in monoecious
plants with separate male and female
flowers (e.g., maize) and in most hermaphroditic animals. Alternatively, male and
female function can be separated in time
within an individual, as found in many
plants (‘‘dichogamy,’’ [23]) and some
animals (‘‘sequential hermaphroditism,’’
[24]); slipper shells, for example, are born
male and become female later in life.
Finally, male and female reproductive
organs can be segregated into different
individuals, as in some plants (such as
papaya and cannabis) and most animals.
Separate sexes have evolved independently many times among plants and
animals, which suggests that there must
be an evolutionary cost to hermaphroditism, at least in some groups. Two major
hypotheses have been proposed to explain
the evolution of separate sexes. The first
hypothesis is that there are trade-offs
between male and female function, such
as when mating displays enhance male
fitness but decrease female fitness. In this
case, individuals can gain reproductive
advantages by specializing as a male or
female [25]. Direct evidence for the tradeoff hypothesis is sparse [26], and observations consistent with it come from hermaphroditic great pond snails, which
reallocate resources to female function
when sperm production is experimentally
abolished [27], and from strawberries, in
which increased pollen production comes
at the cost of reduced seed set [28].
Indirect evidence of a trade-off comes
from the fact that many asexual animals
[29] and plants [30] that still have residual
sperm/pollen production evolve reduced
investment in male gametes over time,
suggesting that doing so increases female
function. The second major hypothesis is
that separate sexes evolve as a means to
avoid self-fertilization, which can produce
low-fitness offspring because of the exposure of recessive deleterious alleles (‘‘inbreeding depression’’) [31]. Empirical
evidence for inbreeding depression is
widespread in animals and plants
[32,33]; for instance, in the Hawaiian
endemic plant genus Scheidia, high inbreeding depression promotes the evolution of dioecy [34].
When separate sexes are favored, the
transition can occur via several evolutionary pathways. Separate sexes may evolve
from hermaphrodites either by gradual
increases in sex-specific investment or
rapidly by the appearance of male- or
female-sterility mutations (Figure 2). The
latter occurs regularly in plants, often
generating mixed sexual systems, such as
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The Myths Deconstructed
These myths do not survive a survey of
sex determination systems across the tree
of life. To deconstruct these myths, we first
provide background on the evolution of
separate sexes. We then summarize the
diversity of sex-determining mechanisms
found among animals and plants and
discuss the evolutionary forces that drive
transitions among systems (Myth 1 revisited). This is followed by a summary of
more recent findings on the underlying
molecular genetics of sex determination
(Myth 2 revisited) and a deconstruction of
common misconceptions of sex chromosome evolution in humans and other
species (Myth 3 revisited). We conclude
with an outlook for future research that
might improve our understanding of how
and why sex determination evolves so
rapidly in many animals and plants.
The Evolution of Separate Sexes
Box 1. From Mating Types to Sexes
Meiotic sex likely has a single origin, which dates back to the origin of eukaryotes
[144,145]). While most eukaryotes display some form of meiotic sex, many lack
differentiated male and female gametes—a situation referred to as isogamy. Even
with isogamy, however, mating is often not random but requires that joining cells
differ at a mating type (MAT) locus. Mating types might have evolved to
orchestrate the developmental transition from the haploid to the diploid phase of
the life cycle [146,147]: plus and minus gametes express complementary
transcription factors, encoded by different alleles at the MAT locus; these
combine in the zygote into heterodimers that silence the genes expressed in the
haploid phase and switch on the diploid program.
Isogamy permits a theoretically unlimited number of mating types; high numbers
increase the probability that randomly mating partners display complementarity.
Most basidiomycete fungi, for instance, present two independent MAT loci (and
are therefore said to be tetrapolar, because a single meiosis can produce cells of
four distinct mating types); each locus can be multiallelic, resulting in up to
thousands of different mating types. Alternatively, a low probability of
encountering complementary partners might have driven a transition to
homothallism observed in many ascomycete fungi, which refers to a mating
compatibility between genetically identical individuals. Homothallism evolved via
genic capture: a single genome harbors complementary mating-type alleles,
which are differentially expressed in plus and minus gametes. Mating-type
switching in yeasts allows different cells from the same clone to express
complementary mating types, and thus enter the diploid phase of their life cycle.
Anisogamy (small male and large female gametes) evolved independently in
many eukaryotic lineages, including several different groups of protists (such as
red algae, brown algae, apicomplexa, dinoflagellates, and ciliates; [148]), as well as
most plants and animals. The transition towards anisogamy is thought to result
from disruptive selection [1,149,150]: given opposing pressures to simultaneously
maximize the number of gametes, their encounter rate, as well as the mass and
ensuing survival of resulting zygotes, the fitness of both partners is often
maximized when one interacting gamete is small and mobile, while its large and
sessile partner provides the resources required for zygote development.
Intermediate gametes do worse than small ones in terms of mobility and
numbers, and worse than large ones in terms of provisioning. Such constraints
largely explain why sexes (at the gametic level) are two and only two, and why
anisogamy independently evolved in many lineages. At the molecular level, one
route to anisogamy is by the incorporation of genes controlling gamete size into
the MAT region [151]. Further extensions of the MAT region, possibly involving
additional sex-antagonistic genes, led to the U and V chromosomes characterizing male and female gametophytes, as found, e.g., in mosses and liverworts
[152].
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Genotypic versus environmental sex
determination
In animals and plants that have evolved
separate sexes, accurate differentiation
With genotypic sex determination (GSD),
which occurs in the majority of species
with known sex-determining mechanisms,
genetic elements specify whether individuals are female or male. In many animals
and some plants, however, the switch to
develop into a female or male does not lie
in the genes. With environmental sex determination (ESD), external stimuli control sex
determination, such as temperature in
reptiles [40], photoperiod in marine amphipods and some barnacles [41,42], and
social factors in many coral-reef-dwelling
fish and limpets [43,44]. Exactly how the
environment triggers sex development has
remained an open question, although a
recent study found that methylation provided the link in European sea bass [45].
In many species, the line between GSD
and ESD is blurred, with certain environments altering the (otherwise genetically
determined) sex of developing offspring
[46]. For example, tongue sole have
differentiated ZW sex chromosomes, but
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Importantly, the evolution of anisogamy does not require the evolution of
separate sexes, because hermaphrodites can produce both sperm and eggs.
Similarly, several unicellular organisms that are anisogamous, such as apixomplexa and dinoflagellates, can make cells that produce sperm as well as cells that
produce eggs. The evolution of completely separate sexes, where individuals
cannot give rise to both sperm and egg descendants, is thought to be fairly
derived and is found primarily among multicellular organisms with rare unicellular
exceptions (e.g., the ciliate Vorticella [153] and several dioecious diatoms [154]).
gynodioecy (mixtures of females and
hermaphrodites) and androdioecy (mixtures of males and hermaphrodites).
Figure 2 highlights the possible pathways
for the evolution of separate sexes from a
hermaphrodite ancestor and illustrates
their relation to sex chromosome evolution. While we have emphasized the
evolutionary transition from hermaphroditism to separate sexes, several examples
are known where the opposite transitions
into fertile males and females is a fundamental developmental process. Contrary
to Myth 1, however, diverse mechanisms
are used to determine sex [3,4] (Figure 3,
Figure 4; Box 2). All crocodiles, most
turtles, and some fish exhibit temperaturedependent sex determination; Wolbachia
infections override existing sex determination systems in many arthropod species
and either kill/sterilize males or transform
them into phenotypic females; male scale
insects eliminate their father’s genome
after fertilization; marine worm Bonellidae
larvae develop as males only if they
encounter a female; and many plants and
animals—including some snails and fish—
change sex during their lifetime in response to environmental or social cues
[3,37].
In fact, sex determination is a rapidly
evolving trait in many lineages (Figure 3),
and sometimes closely related species, or
populations of the same species, have
different modes of sex determination
[3,4,38]. Houseflies, for example, normally have XY sex chromosomes, but dominant masculinizing and feminizing alleles
on other chromosomes exist in some
populations that override sex determination by the XY chromosomes [39]. This
variety has stimulated investigation into
what evolutionary forces drive the turnover of sex determination mechanisms,
what molecular mechanisms underlie the
different modes of sex determination, and
why sex determination is labile in some
taxa and not in others.
occur (e.g., [35,36]), indicating that the
conditions favoring the separation of male
and female function are not always
present.
Myth 1 Revisited—SexDetermining Mechanisms Are
Diverse and Can Evolve Rapidly
Figure 1. Sex chromosome differentiation. A. Reconstructed evolutionary path of sex
chromosome differentiation in humans. Sex chromosomes originate from autosomes that
acquired a sex-determining function (the Sry gene) after their split from monotremes.
Suppression of recombination between the sex chromosomes, associated with degeneration of
the non-recombining region of the Y chromosome, results in the morphological and genetic
differentiation of sex chromosomes. Recombination suppression occurred in multiple episodes
along the human X and Y chromosome, forming so-called evolutionary strata. The oldest stratum
is shared between eutherian mammals and marsupials, while the youngest stratum of humans is
primate-specific. B. The degree of sex chromosome differentiation ranges widely across species,
spanning the entire spectrum of homomorphic to heteromorphic sex chromosomes, from a
single sex-determining locus, as seen in pufferfish, a small differentiated region (strawberry and
emu), most of the sex chromosomes apart from short recombining regions (humans), to the
entire sex chromosome pair, as seen in Drosophila. Note that the sex chromosomes are not drawn
to scale.
doi:10.1371/journal.pbio.1001899.g001
ZW embryos develop into males when
incubated at high temperatures, and sex
reversal is accompanied with substantial
methylation modification of genes in the
sex determination pathway [47].
ESD is favored over GSD when specific
environments are more beneficial to one
sex [3], selecting for sex-determining
mechanisms that match each sex to its
best environment. For example, in some
gobies and wrasses, nest sites are limited,
and a male’s ability to defend his nest
depends on body size; individuals tend to
start life as females, and only become
males once they are sufficiently large to
successfully defend a nesting site [48]. The
reverse transition, from ESD to GSD, is
thought to be favored when the environment is unpredictable or not variable
enough, in which case ESD could produce
strongly skewed sex ratios or intersex
individuals [3]. Indeed, snow skinks, which
are small, live-bearing lizards, have
different sex-determining mechanisms in
different environments. ESD occurs at low
altitudes where early birth is advantageous
for females and the variance in temperature
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between years is low. GSD predominates at
high altitudes where there is no advantage for early-born females and betweenyear variance in temperature is high
[49]. In this situation, ESD produces
optimal sex ratios at low elevations, while
GSD prevents extreme sex ratios at high
altitudes. Importantly, global climate
change poses a threat to species with
temperature-dependent sex determination
if they cannot adapt rapidly enough to
avoid biased sex ratios [50]. Another
threat to ESD systems comes from
within: they may be prone to invasion
by genetic elements that favor biased sex
ratios (see below).
male- or female-biased sex ratios. In
populations with a skewed sex ratio,
selection on autosomal genes typically
favors equal investment in males and
females [51,52], and a new GSD or ESD
system can become established if it restores
a more even sex ratio. An equal number of
males and females is, however, not always
favored, even among autosomal genes
(e.g., with local mate competition, [53]).
In this case, selection for biased sex ratios
can favor the establishment of a new sexdetermining system [54].
Many examples are known of sex
chromosomes, organelles, and endosymbionts that bias the primary sex ratio.
Meiotic drive, where genetic elements bias
the proportion of gametes that carry them,
can create male-biased sex ratios if they
are located on the Y or Z chromosomes (as
seen in many Drosophila species [55]),
whereas driving X or W chromosomes
create female-biased sex ratios (found in D.
simulans [56], stalk-eyed flies [57], and
rodents [58]); autosomal genes that restore
unbiased sex ratios are known in many
systems. Cyto-nuclear conflict arises because cytoplasmic factors such as mitochondria or chloroplast are often inherited
only through the mother, and they favor
production of females, while autosomal
genes are inherited through both sexes and
favor more equal sex ratios. Cytoplasmic
male sterility encoded by mitochondria
has been widely reported in plants,
including maize, petunia, rice, common
bean, and sunflower [59], as have nuclearencoded male fertility restorer genes [60].
Likewise, cellular endosymbionts are only
transmitted through the mother and can
create maternally inherited female-biased
sex ratios; examples include male-killing
bacteria in butterflies and Drosophila
[61,62]. Recurrent invasions of sex ratio
distorters and their suppressors can result
in rapid transitions among sex determination mechanisms between species, and
may be a major force contributing to the
diversity of sex-determining mechanisms
observed across the tree of life.
Turnover of sex chromosomes
More generally, selection on the sex
ratio can trigger transitions between and
among different ESD and GSD systems
[3]. Sex-biased inheritance patterns of
different genetic elements—such as sex
chromosomes, organelles, or endosymbionts—create a conflict over which sex is
preferred, and can drive the evolution of
In species with genotypic sex determination, the chromosome pair that determines sex can change rapidly over time.
Transitions are particularly likely when
the ancestral sex chromosome exhibits
little genetic differentiation, since WW or
YY combinations are then less likely to be
lethal (Figure 5). New sex-determining
genes (or copies of the original gene in a
new location) can lead to transitions within
and between different XY and ZW
systems (Figure 5). Invasions of sex-deter-
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Genomic conflict and transitions in
sex determination
Figure 2. Evolutionary pathways from hermaphroditism to separate sexes. Shown are two-step pathways involving intermediate male- and
female-sterile individuals. Loss-of-function mutations (red) are assumed to be recessive, while gain-of-function mutations (green) are assumed to be
dominant. Ancestral alleles are in black. M, male fertility allele; m, male sterility mutation; F, female fertility allele; f, female sterility mutation. Because
loss of function mutations (red) are almost 50 times more frequent than gain of function mutations (green) in flowering plants, we would expect
pathways 1 (e.g., some poplar species) or 2 (e.g., papaya) to arise earlier. Furthermore, transitions through gynodioecy, pathways 2 and 3 (e.g.,
strawberry) allow females to completely avoid inbreeding depression, while transitions through androdioecy are more costly because males must
compete with hermaphrodites for fertilization and do not have any of their own ovules to fertilize. These theoretical arguments help to account for
the prevalence of gynodioecy and the XY chromosome system (via pathway 2) observed in plants; nevertheless, all four pathways may be biologically
relevant, although no known examples for pathway 4 currently exist.
doi:10.1371/journal.pbio.1001899.g002
mining genes are facilitated when the new
sex-determining gene (or a gene closely
linked to it) has beneficial effects on fitness
[63].
Sexually antagonistic selection, which
occurs when a mutation is beneficial to
one sex but detrimental to the other, can
also drive transitions between sex determination by different pairs of chromosomes [64,65]. For example, if an allele of
an autosomal gene is beneficial to males
but harmful to females and becomes
linked to a dominant masculinizing mutation, then chromosomes that carry both
the male-beneficial and male-dominant
alleles create a novel Y that can replace
the ancestral mechanisms. Conversely,
alleles that benefit females and harm males
can create novel W chromosomes when
linked to feminizing mutations. Turnover
of sex chromosomes can also be triggered
by the degeneration of the Y and W
chromosome, which commonly follows the
cessation of recombination [66,67], and
will result in the replacement of a lowfitness Y or W chromosome with a
nondegenerate one [68].
Sex determination by the whole
genome
In many animals, sex determination
involves the entire genome. With haplodiploidy (found in about 12% of animal
species, including all ants, wasps, and bees)
and paternal genome elimination (found in
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scale insects), males only transmit their
maternal set of genes (see Figure 4; Box 2:
Glossary). The loss of the paternal genome
in sons benefits mothers but not fathers
because these uniparental sons transmit
more of a mother’s genome to grandchildren than do biparental sons [3]. Females
also experience a selective advantage from
haplodiploidy (but not paternal genome
elimination) because unfertilized eggs can
develop and contribute to fitness when
mating opportunities are rare.
the molecular level in D. melanogaster, C.
elegans, and mammals. All three involve a
master-switch
sex-determining
gene,
which led to the birth of Myth 2. Although
the simplicity of a single master-switch is
alluring, this archetype of sex determination is clearly not universal. Below we
discuss systems where sex is determined by
multiple genes, recent molecular data on
the nature and evolution of sex-determining genes, and how sex determination can
vary in different parts of the body.
Polygenic sex determination
Despite numerous theoretical predictions for how and why sex determination
mechanisms change, many hypotheses
remain untested. Only a small proportion
of taxa have actually been characterized
for their sex determination mechanisms,
hindering the use of comparative methods
to assess the factors associated with
transitions between them. However, because sex determination changes so rapidly in many clades, we can catch these
transitions in action to test theoretical
predictions in a direct, experimental
way.
The pathways that control sexual development have been well characterized at
In some species, sex determination is
polygenic. For example, in zebrafish
(Danio rerio), a key developmental model
organism, sex is not controlled by a single
master regulator but is instead a quantitative threshold trait with either a male or
female outcome, which is determined by
multiple regions in the genome [69–71].
While some of those regions contain genes
known to play a role in sex determination
in other organisms [70], there is an
enduring mystery as to how these multiple
loci and the environment interact to
control downstream sexual differentiation in zebrafish. Zebrafish gonads
develop as testes in the absence of
signals from germ line cells, suggesting
that the factors determining sex may
regulate germ cell proliferation [72].
Sex as a threshold trait has been
inferred in several other fish [73–75]
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Myth 2 Revisited—Multiple and
Various Genes Can Determine
Sex
Figure 3. Diversity of sex determination systems for representative plant and animal clades. The bubble insert graph for the plant clades
represents the relative proportion of species with documented sex chromosomes within plants with separate sexes. Vertebrates: Mammalia
(placental, marsupial, and monotreme mammals), Aves (birds), Reptilia (turtles, snakes, crocodiles, lizards), Amphibia (frogs, toads, salamanders), and
Teleostei (bony fishes). Invertebrates: Acari (mites and ticks), Crustacea (shrimps, barnacles, crabs), and Insects, which include Coccoidea (scale
insects), Coleoptera (beetles), Hymenoptera (ants, bees, and wasps), Lepidoptera (butterflies), and Diptera (flies). Plants: Gymnosperms (non-flowering
plants) and Angiosperms (flowering plants).
doi:10.1371/journal.pbio.1001899.g003
and invertebrates [76], and further
examples of multiple interacting loci
controlling sex determination are no
doubt waiting to be described. Indeed,
in taxa where separate sexes evolved
recently from a hermaphrodite ancestor,
as is common in plants, multiple sexdetermining loci are in fact expected,
since at least two independent mutations—one suppressing male function,
one suppressing female function—are
necessary to produce separate sexes
from a hermaphrodite (Figure 2). If
separate sexes evolve by gradual increase in sexual investment from a
hermaphrodite, sex determination may
also be due to polygenic inheritance.
The nature and evolution of sexdetermining genes and pathways
Some taxa have master-switch sexdetermining genes that are highly conserved, such as the Sry gene in nearly all
mammals [77]. In other lineages, such as
fish from the genus Oryzias [78–80], the
master-switch gene differs among closelyrelated species (Table 1). There is some
empirical evidence for the repeated use of
the same master sex determination switch
genes in animals. For example, in vertebrates other than mammals, dmrt1 (a DM
family gene) and its paralogs act as the
primary sex determination signal in African clawed frog (Xenopus laevis) [13],
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chicken (Gallus gallus) [12], medaka fish
(Oryzias latipes) [78,79], and possibly the
smooth tongue sole (Cynoglossus semilaevis)
[14]. In insects, paralogs of transformer (tra),
a key gene in the sex determination
cascade of Drosophila, have evolved as the
primary switch in houseflies Musca domestica [17], as well as the haplodiploid wasp
Nasonia vitripennis [15] and the honeybee
Apis mellifera [16].
These data suggest that there are
constraints on the types of genes that can
be co-opted as master sex determination
genes [81]. Nevertheless, there are several
cases of switch genes with no homologs in
closely related taxa. These include an
immunity-related gene in rainbow trout
(Oncorhynchus mykiss) [82] and Sxl in
Drosophila [83], whose ortholog has a
non-sex-related function in mRNA splicing in other flies [84]. The primary
master sex-determining gene in the
silkworm Bombyx mori is a W-derived
female-specific piRNA (produced from a
piRNA precursor termed Fem) that
targets a Z-linked gene (named Masc),
and silencing of Masc mRNA by Fem
piRNA is required for female development [85]. Undoubtedly, many other sex
determination genes remain to be found,
making it unclear at present whether
there truly are constraints on the types of
genes that could evolve to be master
control switches.
No master sex determination gene has
been identified in dioecious plants, but
genes that affect flower sex determination
have been found [86,87]. Indeed, many
genes may serve as potential targets for sex
determination in plants, given that male or
female sterility can evolve in various ways
[86]. For example, 227 male-sterility genes
have been identified in rice, with at least
one male-sterility gene found on each of
rice’s 12 chromosomes—hence each autosome could, in principle, evolve a sexdetermining function [88]. This abundance and diversity within a single species
indicates that the initial evolution of
separate sexes is unlikely to be limited to
a scant handful of master genes.
In sharp contrast with the diversity of
primary sex-determining signals, some
key regulatory genes play conserved
roles in the molecular pathways leading
to male or female gonad development
across invertebrates and vertebrates,
such as the doublesex-mab3 (DM) family
genes [89,90]. Despite profound differences in the mode of sex determination
and the identity of the master-switch
genes, DM genes are specifically expressed in the developing gonads of
almost all animals, including vertebrates
(mammals [91], birds [92], turtles and
alligators [93–95], amphibians [96], and
fish [97]) and invertebrates (Drosophila
[98], hymenoptera [99], crustaceans
6
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Figure 4. Schematic overview of some sex determination (SD) mechanisms. M refers to meiosis, F to fertilization. Haploid stages (n) are
indicated as shaded areas and diploid stages (nn) are unshaded. Hermaphrodites: Most flowering plants (and gastropods and earthworms)
simultaneously contain both male and female sexual organs (simultaneous hermaphrodites). Many fish and some gastropods and plants are sequential
hermaphrodites; clownfish, for example, are born males and change into females, while many wrasses or gobies begin life as females and then change
to males. Environmental Sex Determination: In turtles and some other reptiles, sex is determined by incubation temperature of the eggs
(temperature-dependent sex determination). Social factors can act as primary sex-determining cues: sexually undifferentiated larvae of the marine
green spoonworm that land on unoccupied sea floor develop into females (and grow up to 15 cm long), while larvae that come into contact with
females develop into tiny males (1–3 mm long) that live inside the female. Genotypic Sex Determination: Almost all mammals and beetles, many
flies and some fish have male heterogamety (XY sex chromosomes), while female heterogamety (ZW sex chromosomes) occurs in birds, snakes,
butterflies, and some fish. In mosses or liverworts, separate sexes are only found in the haploid phase of the life cycle of an individual (UV sex
chromosomes). In some flowering plants and fish, such as zebrafish, sex is determined by multiple genes (polygenic sex determination). In bees, ants,
and wasps, males develop from unfertilized haploid eggs, and females from fertilized diploid eggs (haplodiploidy), while males of many scale insects
inactivate or lose their paternal chromosomes (paternal genome elimination). In some species, sex is under the control of cytoplasmic elements, such
as intracellular parasites (e.g., Wolbachia) in many insects or mitochondria in many flowering plants (cytoplasmic sex determination). In some flies and
crustaceans, all offspring of a particular individual female are either exclusively male or exclusively female (monogeny).
doi:10.1371/journal.pbio.1001899.g004
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[100,101], and mollusks [102,103]).
Thus, the evolution of sex-determining
pathways, at least in animals, appears to
occur by the recruitment of new masterswitches controlling sexual fate, while
the downstream developmental pathways that regulate gonadal differentiation are retained [10,81,104], although
the function of some of these downstream elements appears to diverge
among lineages [105]. Characterization
of polygenic sex determination systems
and identification of master sex determination genes across kingdoms will provide insight into the mechanistic constraints limiting the evolution of sex
determination pathways.
Sex determination: soma vs. germ
line
Sex determination can also differ with
respect to where in the body sex is
determined. In humans, sex is determined
in the developing gonad, and gonadal sex
hormones in turn trigger sex determination and differentiation in nongonadal
tissues. By contrast, in birds, Drosophila,
and nematodes [106–109], sexual differentiation is a cell-autonomous process,
although secreted signaling molecules can
play a role in generating sexual dimor-
phism in somatic tissues. Studies in Drosophila have shown that only a subset of cells
express the genes of the sex determination
cascade and have a sexual identity [106].
Cell-autonomous sex determination can
result in the formation of gynandromorphs—individuals that contain both
male and female characteristics, found in
birds and many insects, including butterflies and beetles. Sex determination can
also be regulated differently in the soma
versus the germ line of the same species
[110,111]. In houseflies [112] and some
frogs [113] and fish [114–116], transplantation experiments indicate that the sex of
germ cells depends on the surrounding
soma, i.e., XX germ cells transplanted into
male soma form sperm, and XY germ cells
transplanted in a female soma form oocytes. In contrast, germ cells in Drosophila
[117] and mammals [118] receive signals
from the surrounding somatic gonad, but
they also make an autonomous decision
during germ line sexual development; this
may also be true for chickens [107]. In
these animals, the ‘‘sex’’ of the soma must
match the ‘‘sex’’ of the germ cells for proper
gametogenesis to occur. If XX germ cells
are transplanted into male soma they do
not form sperm, and XY germ cells
transplanted into female soma fail to form
oocytes.
Myth 3 Revisited—Sex
Chromosomes’ Eternal Youth
Heteromorphic
sex
chromosomes
evolve from autosomes that are initially
identical but then stop recombining and
differentiate. Recombination suppression
is favored when it links together sexually
antagonistic alleles and sex-determining
loci (i.e., a male-beneficial allele and a
male-determining gene on a Y chromosome, or a female-beneficial allele and a
female-determining gene on a W chromosome). A side effect of repressed recombination on Y and W chromosomes is that
natural selection is inefficient (reviewed in
[4,5]), which can result in the loss of most
of their genes. Y or W degeneration has
occurred in many animal taxa with
heteromorphic sex chromosomes, including mammals [119], many birds [120],
snakes [121], and many insects [122,123],
along with some plants, including Rumex
[124]. In the most extreme cases, the Y or
W is entirely lost, resulting in so-called X0
and Z0 systems. According to Myth 3,
differentiation of sex chromosomes is
evolutionarily inevitable, and the degree
of heteromorphism reflects their age
(Figure 5). However, as we explain below,
evidence from a broad array of organisms
indicates that the link between sex chro-
Figure 5. Transitions versus differentiation of sex chromosomes. Transitions between homomorphic sex chromosomes result from new
masculinizing (M9) or feminizing (F9) mutations that invade an existing XY or ZW system to create a new chromosome pair (in grey) that harbors the
sex-determining gene (additional transitional karyotypes are indicated by unshaded circles). XYRXY transitions result in the loss of the ancestral Y
(and ZWRZW transitions cause loss of the ancestral W). Transitions between XY and ZW systems result in some offspring that are homozygous for
the Y (blue) or W (red) chromosome and are thus more likely if the chromosomes have similar gene content but become increasingly difficult if these
chromosomes have degenerated (side boxes on left and right), causing YY and WW individuals to be less fit.
doi:10.1371/journal.pbio.1001899.g005
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Box 2. Glossary of Sex-Determining Mechanisms
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Hermaphrodites: individuals that contain both male and female sex organs.
Simultaneous hermaphroditism: male and female sexual organs coexist in one individual (e.g., most flowering plants and
earthworms, many terrestrial gastropods).
Sequential hermaphroditism: individuals change sex at some point during their life (e.g., many fish, snails, and some plants).
Dioecy (plants) or gonochorism (animals): individuals are either male or female throughout their life.
Environmental sex determination: sex is triggered by environmental cues, such as temperature, pH, social interactions, and
seasonality (e.g., many reptiles and some fish).
Genotypic sex determination: an individual’s sex is established by its genotype (e.g., mammals, birds, amphibians, most
insects, some reptiles and fish, and some plants).
Male heterogamety: type of genotypic sex determination in which males are heterozygous for the sex-determining locus
(termed X and Y, as seen in therian mammals and Drosophila).
Female heterogamety: type of genotypic sex determination in which females are heterozygous for the sex-determining locus
(termed Z and W, as seen in birds, snakes, butterflies, and gingko trees).
UV sex determination: separate sexes are only found in the haploid phase of the life cycle of an individual (e.g., mosses or
liverworts).
Polygenic sex determination: sex is determined by multiple genes (e.g., some fish and flowering plants).
Haplodiploidy: males develop from unfertilized, haploid eggs, and females from fertilized, diploid eggs (e.g., bees, ants, and
wasps).
Paternal genome elimination: paternal chromosomes in males are inactivated or lost after fertilization, leaving males
functionally haploid (e.g., many scale insects).
Cytoplasmic sex determination: sex is under the control of cytoplasmic elements, such as intracellular parasites (e.g.,
Wolbachia in many insects) or mitochondria (e.g., cytoplasmic male sterility in flowering plants).
Monogeny: all offspring of a particular individual female are either exclusively male or exclusively female (e.g., some flies and
crustaceans).
Sexual reproduction: the mixing of genomes via meiosis and fusion of gametes.
Sex: the sexual phenotype of an individual.
Sex determination: the mechanism by which the sexual phenotype of an individual is established in a given species.
Sex chromosome: a chromosome involved with determining the sex of an individual.
Autosome: a chromosome not involved with determining the sex of an individual (i.e. any chromosome that is not a sex
chromosome).
Y degeneration: the loss of genetic information on the non-recombining Y chromosome.
Homomorphic sex chromosomes: sex chromosomes that are morphologically indistinguishable.
Heteromorphic sex chromosomes: sex chromosomes that are morphologically distinct.
Sexually antagonistic selection: selection for a trait that benefits one sex to the detriment of the other sex.
Gynodioecy: a breeding system that consists of a mixture of females and hermaphrodites.
Androdioecy: a breeding system that consists of a mixture of males and hermaphrodites.
Meiotic drive (also called segregation distortion): a system in which genetic elements termed segregation distorters bias the
proportion of gametes that carry them, resulting in over- or under-representation of one gametic type (i.e. non-mendelian
segregation).
Nucleo-cytoplasmic conflict: conflict in inheritance patterns between the nuclear genome and organelle genomes that are
transmitted only maternally.
Gynandromorphs: individuals that contain both male and female characteristics.
mosome heteromorphism and age is often
far from direct.
Not all sex chromosomes become
differentiated
Differentiation is often seen as the
default path of sex chromosome evolution,
but contrary to Myth 3, some ancient sex
chromosomes recombine and are undifferentiated over most of their length.
Examples are found in python snakes
and ratite birds, whose homomorphic sex
chromosomes are about 140 and 120
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million
years
old,
respectively
[121,125,126], i.e. almost as old as the
heteromorphic sex chromosomes of therian mammals (about 180 million years old).
How do some ancient sex chromosomes
avoid differentiation? One hypothesis is
that occasional X-Y recombination purges
deleterious alleles on the Y. A mechanism
proposed for tree frogs is that XY embryos
are occasionally sex-reversed, and so the X
and Y recombine when these embryos
develop into females [127,128]. Second,
some taxa may have few genes under
sexually antagonistic selection on their sex
chromosomes and thus avoid selection to
suppress recombination between the X
and Y [129]. Third, sexually antagonistic
selection can be resolved by other means,
such as the evolution of sex-specific
expression [130]. Sexually antagonistic
alleles can accumulate along the sex
chromosomes, and sex-specific expression
will confine the product of such alleles to
the sex they benefit, thereby eliminating
the selective pressure for recombination
suppression. Consistent with this last
9
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Table 1. Known master sex-determining genes in vertebrates and insects, and their paralogs.
Species
Master sex
determining gene
Sex-determining
mechanisms
Gene paralog
Paralog function
Reference
mammals
Sry
sex-determining Y
Sox3
HMG-box
transcription factor
[77]
chicken (Gallus gallus)
dmrt1
dose-dependent Z
-
SD pathway
transcription factor
[12]
African clawed frog
(Xenopus laevis)
dmW
sex-determining W
dmrt1
SD pathway
transcription factor
[13]
medaka (Oryzias latipes)
dmrt1Y
sex-determining Y
dmrt1
SD pathway
transcription factor
[78,79]
(Oryzias luzonensis)
gsdfY
sex-determining Y
gsdf
secretory protein in
SD pathway
[80]
Patagonian pejerrey
(Odontesthes hatcheri)
amhY
sex-determining Y
amh
anti-Mullerian hormone
[155]
rainbow trout
(Oncorhynchus mykiss)
sdY
sex-determining Y
Irf9
interferon
regulatory factor
[82]
tiger pufferfish (Takifugu
rubripes)
amhr2
dose-dependent X
amhr
anti-Mullerian
hormone receptor
[156]
smooth tongue sole
(Cynoglossus semilaevis)
dmrt1
dose-dependent Z
-
SD pathway
[14]
fruit flies (Drosophila)
Sxl
dose-dependent X
CG3056
mRNA splicing,
non-sex specific
[83,84]
housefly (Musca domestica)
F
sex-determining W
tra
SD pathway switch
splice factor
[17]
silkworm (Bombyx mori)
Fem
sex-determining W
-
piRNA
[85]
honeybee (Apis mellifera)
csd
haplodiploid
tra
SD pathway switch
splice factor
[16]
wasp (Nasonia vitripennis)
Nvtra
haplodiploid
tra
SD pathway
switch splice factor
[15]
doi:10.1371/journal.pbio.1001899.t001
possibility, the recombining, non-differentiated region along the sex chromosomes
of the emu (a ratite bird) contains an
excess of genes whose expression is sexbiased, relative to autosomes [126].
Y chromosomes are not doomed
Y chromosome degeneration has
prompted the suggestion that the human
Y will eventually disappear [131–133], a
claim based on the naı̈ve assumption of a
constant rate of gene loss from the Y over
time. However, theory predicts that the
rate of gene decay on the Y decreases over
evolutionary time and should halt on an
old, gene-poor Y chromosome [67,134].
Recent comparative genomic studies support this hypothesis as the gene content of
the primate Y chromosome has been
stable over the last 25 million years,
suggesting that an equilibrium gene content has been reached in humans [135].
Moreover, old gene-poor Y chromosomes
that are tens of millions of years old, such
as the Drosophila Y [136], actually show a
net rate of gene gain rather than gene loss
[137]. Thus, the Y chromosome can be a
stable and important component of the
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genome in many species, and may even
prevent turnover of sex-determining
mechanisms (see below).
In contrast to the lability of sex
determination mechanisms in some
groups, eutherian mammals, birds and
many insects exhibit virtually no variation
in how sex is determined (Figure 3). This
stability could be due to an absence of
genetic variation, particularly when multiple genetic steps are required for a
transition to a new sex-determining system
(Figure 2). Mutations are known, however,
that override sex determination (Table 1)
[138], suggesting that the conservation is
not due to a lack of genetic variation.
Instead, evolutionary traps may stabilize
sex-determining systems for long spans of
evolutionary time.
Heteromorphic sex chromosomes may
act as just such a trap. Transitions between
XY and ZW systems that create YY or
WW individuals are prevented when Y or
W chromosomes lack essential genes
(Figure 5). Also, if the Y (or W) chromosome
has evolved sex-essential genes, such as
spermatogenesis genes located on the
human and Drosophila Y, sex chromosome transitions are only possible if these
genes are moved to another chromosome, since the old Y, along with its
genes, is lost during the transition
(Figure 5). Overall, phylogenetic patterns
in vertebrates or insects [3,139] are
consistent with the notion that heteromorphic sex chromosomes constrain
shifts in sex determination mechanism,
but several notable exceptions exist in
both groups. In rodents, for example,
many species with unusual sex-determining systems can be found: XY females in
some lemming species, X0 females or
XX males in vole species, and X0
females and males in some Japanese
spiny rats and mole voles [140]. Likewise, some insect groups are known that
harbor variation in sex chromosome
karyotype among species; in grasshoppers, fusions between sex-chromosomes
and autosomes combined with Y-degeneration and/or Y-loss have created much
variation in sex chromosome karyotype,
including species with multiple X or Y
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Evolutionary traps and conserved
sex-determining systems
chromosomes [141]; true fruit flies (Tephritidae) that contain both XY and ZW
species [142]; or blowfly species that
have secondarily lost their heteromorphic
sex chromosomes [143].
How much sex chromosome heteromorphism is required to create a trap, and
how strong this trap is, remains unknown.
To date, only one example of the reversal
of an ancient sex chromosome back to an
autosome has been characterized. Specifically, all Drosophila species contain an
autosome that was formerly an X chromosome: the dot chromosome. This
chromosome still has a minor feminizing
role during sex determination, is targeted
by a chromosome-specific regulatory
mechanism similar to dosage compensation of the X, and its patterns of biased
gene expression during early embryogenesis, oogenesis, and spermatogenesis resemble that of the current X in Drosophila
[136]. The retention of the specialized
genomic architecture of highly differentiated sex chromosomes on the dot chromosome illustrates the numerous barriers
to sex chromosome turnover that exist in
highly heteromorphic systems, even
though there are some cases where these
are overcome.
Haplodiploidy also appears to be an
evolutionary trap. While it has arisen a few
dozen times, the reverse transition has not
been reported [3]. Transitions from or to
haplodiploidy require changes in genetic
architecture and meiotic mechanisms,
which are likely more complex than a
simple change in a master-switch sexdetermining gene. Furthermore, females
switching from haplodiploidy would lose
the fitness benefit associated with producing uniparental sons.
Systems that involve interacting
somatic and germ line sex determination
mechanisms may also limit transitions of
sex-determining mechanisms, since changes in either germ line sex or somatic sex
alone may produce infertile individuals
[111]. Thus, while sex determination is
generally characterized by diversity and
turnover, some sex-determining systems
appear to be more evolutionarily stable
than others [3].
Outlook
Studying the forces that drive the evolution of sex determination has mainly come
from theoretical works, with little empirical
data. However, the genomic revolution has
allowed researchers to address scientific
questions and tackle novel biological systems
at the molecular level. As new genomic
approaches increase the pace of discovery
and characterization of sex determination
innon-model organisms, we anticipate that
comparative phylogenetic methods will be
key to examining the roles of various
ecological and genetic factors that drive
changes in sex determination mechanisms.
Additionally, genomic data make it increasingly possible to map sex-determining loci
from closely related species and to identify
the evolutionary mechanisms hypothesized
to cause transitions among sex-determining
systems. Finally, comparative and functional
genomic data will allow researchers to
address how new master sex determination
genes are incorporated into existing genetic
networks controlling sexual development. A
full understanding of the diversity of sex
determination mechanisms will require that
we expand the taxonomic breadth of study
systems well beyond classic model organisms. Promising models include dipteran
insects, such as houseflies or chironomids;
teleost fish; and reptilian clades, including
turtles and lizards; as well as plant genera,
such as strawberries, that show variation
within and between species in how sex
(or gender in plants) is determined. Integrative and interdisciplinary approaches across
the tree of life will illuminate the diversity of
sex determination and yield exciting new
insights of how and why sex determination
evolves in animals and plants.
Acknowledgments
Membership of the Tree of Sex Consortium
(http://www.treeofsex.org/): Doris Bachtrog,
Judith E. Mank, Catherine L. Peichel, TiaLynn Ashman, Heath Blackmon, Emma E.
Goldberg, Matthew W. Hahn, Mark Kirkpatrick, Jun Kitano, Itay Mayrose, Ray Ming,
Sarah P. Otto, Matthew W. Pennell, Nicolas
Perrin, Laura Ross, Nicole Valenzuela, Jana C.
Vamosi.
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